EP2194064A1 - FGF-21 fusion proteins - Google Patents

FGF-21 fusion proteins Download PDF

Info

Publication number
EP2194064A1
EP2194064A1 EP09150627A EP09150627A EP2194064A1 EP 2194064 A1 EP2194064 A1 EP 2194064A1 EP 09150627 A EP09150627 A EP 09150627A EP 09150627 A EP09150627 A EP 09150627A EP 2194064 A1 EP2194064 A1 EP 2194064A1
Authority
EP
European Patent Office
Prior art keywords
fgf
gly
igg4
terminus
fusion protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09150627A
Other languages
German (de)
English (en)
French (fr)
Inventor
Wolfgang Glaesner
Rohn Lee Millican, Jr
Yu Tian
Sheng-Hung Rainbow Tschang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP2194064A1 publication Critical patent/EP2194064A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to fibroblast growth factor 21 compounds fused to proteins that have the effect of extending the in vivo half-life of the polypeptides. These fusion proteins can be used to treat non-insulin dependent diabetes mellitus, obesity and metabolic syndrome.
  • Fibroblast growth factors are large polypeptides widely expressed in developing and adult tissues ( Baird et al., Cancer Cells, 3:239-243, 1991 ) and play crucial roles in multiple physiological functions including angiogenesis, mitogenesis, pattern formation, cellular differentiation, metabolic regulation and repair of tissue injury ( McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998 ). According to the published literature, the FGF family now consists of twenty-two members ( Reuss et al., Cell Tissue Res. 313:139-157 (2003 )).
  • Fibroblast growth factor 21 has been reported to be preferentially expressed in the liver and described as a treatment for ischemic vascular disease, wound healing, and diseases associated with loss of pulmonary, bronchia or alveolar cell function and numerous other disorders ( Nishimura et al., Biochimica et Biophysica Acta, 1492:203-206, (2000 ); US 6,716,626 and WO01/18172 ).
  • FGF-21 has been shown to stimulate glucose-uptake in mouse 3T3-L1 adipocytes in the presence or absence of insulin, and to decrease fed and fasting blood glucose levels in ob / ob and db / db mice and 8 week old ZDF rats in a dose-dependant manner, thus, providing the basis for the use of FGF-21 as a therapy for treating type 2 diabetes and obesity ( WO03/011213 ).
  • the present invention is based on the finding that the fusion of a protein with a long circulating half-life, such as the Fc portion or an immunoglobulin or albumin, to a FGF-21 compound results in a biologically active, FGF-21 fusion protein with an extended elimination half-life and reduced clearance when compared to that of native FGF-21.
  • the FGF-21 fusion proteins of the present invention have greater usefulness as a therapeutic as well as greater convenience of use than wild-type FGF-21 because they retain all or a portion of the biological activity of wild-type FGF-21 yet have an extended time action when compared to that of the wild-type FGF-21.
  • FGF-21 fusion proteins of the present invention are useful to treat subjects with disorders including, but not limited to, type 2 diabetes, obesity, and metabolic syndrome, with particular advantages being that the FGF-21 fusion proteins of the present invention have improved efficacy due to constant exposure and require fewer doses, increasing both the convenience to a subject in need of such therapy and the likelihood of a subject's compliance with dosing requirements.
  • Compounds of the present invention include a heterologous fusion protein comprising a first polypeptide with a N-terminus and a C-terminus fused to a second polypeptide with a N-terminus and a C-terminus wherein the first polypeptide is a FGF-21 compound and the second polypeptide is selected from the group consisting of
  • the present invention also includes polynucleotides encoding the heterologous fusion protein described herein, vectors comprising these polynucleotides and host cells transfected or transformed with the vectors described herein. Also included is a process for producing a heterologous fusion protein comprising the steps of transcribing and translating a polynucleotide described herein under conditions wherein the heterologous fusion protein is expressed in detectable amounts.
  • Another embodiment of the present invention encompasses pharmaceutical compositions of FGF-21 fusion proteins and methods of treating a patient suffering from type 2 diabetes, obesity, or metabolic syndrome comprising administering to said patient a therapeutically effective amount of a heterologous fusion protein described herein.
  • FGF-21 is a 208 amino acid polypeptide containing a 27 amino acid leader sequence.
  • Human FGF-21 has ⁇ 79% amino acid identity to mouse FGF-21 and ⁇ 80% amino acid identity to rat FGF-21.
  • Human FGF-21 or a mutein thereof is the preferred polypeptide template for the FGF-21 fusion proteins of the present invention but it is recognized that one with skill in the art could readily make fusion proteins based on an alternative mammalian FGF-21 polypeptide sequence.
  • amino acid positions of the present invention are determined from the mature, wild type or native human 181 amino acid FGF-21 polypeptide as shown below (SEQ ID NO: 1):
  • the corresponding DNA sequence coding for the mature human 181 amino acid FGF-21 polypeptide is (SEQ ID NO:2):
  • the FGF-21 useful in the methods of the present invention is preferably a mutein, analog or derivative of human FGF-21 as shown in SEQ ID NO: 1, hereinafter collectively known as FGF-21 compounds.
  • FGF-21 compounds have sufficient homology to FGF-21 such that the compounds have the ability to bind to the FGF-21 receptor and initiate a signal transduction pathway resulting in glucose uptake stimulation or other physiological effects as described herein.
  • FGF-21 compounds can be tested for glucose uptake activity using a cell-based assay such as that described in Example 1.
  • a “subject” or “patient” is a mammal, preferably a human.
  • Type 2 diabetes non-insulin dependent diabetes mellitus (NIDDM)
  • NIDDM non-insulin dependent diabetes mellitus
  • Glucose intolerance can be defined as an exceptional sensitivity to glucose.
  • Hyperglycemia is defined as an excess of sugar (glucose) in the blood.
  • hypoglycemia also called low blood sugar, occurs when your blood glucose level drops too low to provide enough energy for your body's activities.
  • Hyperinsulinemia is defined as a higher-than-normal level of insulin in the blood.
  • Insulin resistance is defined as a state in which a normal amount of insulin produces a subnormal biologic response.
  • Metabolic syndrome can be defined as a cluster of at least three of the following signs: abdominal fat - in most men, a 40-inch waist or greater; high blood sugar - at least 110 milligrams per deciliter (mg/dl) after fasting; high triglycerides - at least 150 mg/dL in the bloodstream; low HDL - less than 40 mg/dl; and, blood pressure of 130/85 or higher.
  • Native or wild-type refers to the mature human 181 amino acid FGF-21 polypeptide as shown in SEQ ID NO: 1.
  • the term "native” or “wild-type” is intended to encompass allelic variants of the polypeptide in question.
  • amino acid is used herein in its broadest sense, and includes naturally occurring amino acids as well as non-naturally occurring amino acids, including amino acid variants and derivatives.
  • amino acid variants and derivatives include, for example, naturally occurring proteogenic L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non-proteogenic amino acids such as norleucine, ⁇ -alanine, ornithine, etc.; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids.
  • non-naturally occurring amino acids include ⁇ -methyl amino acids (e.g.
  • the FGF-21 compounds of the present invention comprise only naturally occurring amino acids except as otherwise specifically provided herein.
  • In vitro potency is the measure of glucose uptake of a FGF-21 fusion protein in a cell-based assay and is a measure of the biological potency of a FGF-21 compound. In vitro potency is expressed as the "EC 50 " which is the effective concentration of compound that results in 50% activity in a single dose-response experiment. For the purposes of the present invention, in vitro potency is determined using a glucose uptake assay that employs 3T3-L1 cells (Example 1).
  • plasma half-life refers to the time in which half of the relevant molecules circulate in the plasma prior to being cleared.
  • An alternatively used term is "elimination half-life.”
  • extended time action or “longer time action” used in the context of plasma half-life or elimination half-life indicates there is a statistically significant increase in the half-life of a FGF-21 fusion protein relative to that of the reference molecule (e.g., the non-fusion form of the polypeptide or the native polypeptide) as determined under comparable conditions.
  • a FGF-21 fusion protein of the present invention has an elimination half-life greater than that of a comparable FGF-21 compound.
  • the half-life reported herein in Example 3 is the elimination half-life; it is that which corresponds to the terminal log-linear rate of elimination. Those of skill in the art appreciate that half-life is a derived parameter that changes as a function of both clearance and volume of distribution.
  • Clearance is the measure of the body's ability to eliminate a drug. As clearance decreases due, for example, to modifications to a drug, half-life would be expected to increase. However, this reciprocal relationship is exact only when there is no change in the volume of distribution. A useful approximate relationship between the terminal log-linear half-life (t 1 ⁇ 2), clearance (C), and volume of distribution (V) is given by the equation: t 1 ⁇ 2 ⁇ 0.693 (V/C). Clearance does not indicate how much drug is being removed but, rather, the volume of biological fluid such as blood or plasma that would have to be completely freed of drug to account for the elimination. Clearance is expressed as a volume per unit of time (See Example 3).
  • the heterologous fusion proteins of the present invention comprise a FGF-21 compound fused to the Fc portion of an immunoglobulin, an analog of the Fc portion of an immunoglobulin, a fragment of the Fc portion of an immunoglobulin, human albumin, a human albumin analog, or a human albumin fragment.
  • the C-terminus of the FGF-21 compound may be fused directly, or fused via a peptide linker, to the N-terminus of an albumin or Fc protein.
  • the N-terminus of the FGF-21 compound may be fused directly, or fused via a peptide linker, to the C-terminus of an albumin or Fc protein.
  • a "human FGF-21 mutein” is defined as comprising human FGF-21 in which at least one amino acid of the wild-type mature protein has been substituted by another amino acid.
  • FGF-21 muteins are described in U.S. patent applications 60/528,582 , 60/606,805 , 60/606,830 , and 60/635,882 herein incorporated by reference.
  • a mutein possesses some modified property, structural or functional, of the wild-type protein.
  • the mutein may have enhanced or improved physical stability in concentrated solutions (e.g ., less hydrophobic mediated aggregation), while maintaining a favorable bioactivity profile.
  • the mutein may possess increased compatibility with pharmaceutical preservatives (e.g ., m -cresol, phenol, benzyl alcohol), thus enabling the preparation of a preserved pharmaceutical formulation that maintains the physiochemical properties and biological activity of the protein during storage.
  • pharmaceutical preservatives e.g ., m -cresol, phenol, benzyl alcohol
  • the mutein may have reduced O-glycosylation when expressed in yeast.
  • the mutein may have less deamindation when compared to wild type FGF-21. As used herein, these terms are not limiting, it being entirely possible that a given mutein has one or more modified properties of the wild-type protein.
  • FGF-21 muteins with enhanced pharmaceutical stability include the substitution with a charged and/or polar but uncharged amino acid for one or more of the following: glycine 42, glutamine 54, arginine 77, alanine 81, leucine 86, phenylalanine 88, lysine 122, histidine 125, arginine 126, proline 130, arginine 131, leucine 139, alanine145, leucine 146, isoleucine 152, alanine 154, glutamine 156, glycine 161, serine 163, glycine 170, or serine 172 wherein the numbering of the amino acids is based on SEQ ID NO:1.
  • a charged amino acid is defined as a positively or negatively charged amino acid.
  • a positively charged amino acid is defined to include histadine, lysine, arginine, and non-naturally occurring analogs thereof (e.g. , gamma aminobutyric acid, ornithine, etc .).
  • a negatively charged amino acid is defined to included aspartate, glutamate, and non-naturally occurring analogs thereof (e.g. , aminoadipic acid).
  • a polar but uncharged amino acid is defined to include serine, threonine, asparagine, glutamine, and non-naturally occurring analogs thereof.
  • Preferred muteins are Gln54Glu, Leu139Glu, Ala145Glu, Leu146Glu, Ile152Glu, Gln156Glu, Ser163Glu, and He152Glu-Ser163Glu.
  • FGF-21 with enhanced pharmaceutical stability include FGF-21 with the substitution of a cysteine for two or more of the following: arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27, lutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58, valine 62, leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine 73, glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81, leucine 82, glycine 84, serine 85, proline 90, alanine 92, serine 94, phenylalanine 95, leucine 100, aspartate
  • Preferred muteins with engineered disulfide bonds are Tyr22Cys-Leu139Cys; Asp24Cys-Arg135Cys; Leul18Cys-Glyl32Cys; His117Cys-Pro130Cys; His17Cys-Ala129Cys; Leu82Cys-Pro119Cys; Gly80Cys-Ala129Cys; Gly43Cys-Pro124Cys; Gly42Cys-Arg126Cys; Gly42Cys-Pro124Cys; Gln28Cys-Pro124Cys; Gln27Cys-Ser123Cys; Ala26Cys-Lys122Cys; or Asp25Cys-Lys122Cys.
  • Most preferred muteins with engineered disulfide bonds are Leu118Cys-Ala134Cys; Leu21Cys-Leu33Cys; Ala26Cys-Lys122Cys; Leu21Cys-Leu33Cys/Leu118Cys-Ala134Cys; Ile152Glu-Ser163Glu/Leu118Cys-Ala134Cys; and, Leu118Cys-Ala134Cys/Ser167Ala.
  • Examples of muteins of human FGF-21, or a biologically active peptide thereof, with reduced capacity of O-glycosylation when expressed in yeast compared to wild-type human FGF-21 include the substitution of any amino acid except Ser or Thr for Ser 167, in combination with the substitution of a cysteine for two or more of the following: arginine 19, tyrosine 20, leucine 21, tyrosine 22, threonine 23, aspartate 24, aspartate 25, alanine 26, glutamine 27, glutamine 28, alanine 31, leucine 33, isoleucine 35, leucine 37, valine 41, glycine 42, glycine 43, glutamate 50, glutamine 54, leucine 58, valine 62, leucine 66, glycine 67, lysine 69, arginine 72, phenylalanine 73, glutamine 76, arginine 77, aspartate 79, glycine 80, alanine 81, le
  • the most preferred muteins with reduced capacity of O-glycosylation when expressed in yeast compared to wild-type human FGF-21 are Leu118Cys-Ala134Cys-Ser167Ala; Leu21Cys-Leu33Cys-Ser167Ala; Ala26Cys-Lys122Cys-Ser167Ala; or Leu21Cys-Leu33Cys/Leu118Cys-Ala134Cys-Ser167Ala.
  • An FGF-21 compound also includes an "FGF-21 derivative" which is defined as a molecule having the amino acid sequence of FGF-21 or an FGF-21 analog, but additionally having a chemical modification of one or more of its amino acid side groups, ⁇ -carbon atoms, terminal amino group, or terminal carboxylic acid group.
  • a chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties.
  • Modifications at amino acid side groups include, without limitation, acylation of lysine ⁇ -amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine.
  • Modifications of the terminal amino group include, without limitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications.
  • Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications.
  • one or more side groups, or terminal groups may be protected by protective groups known to the ordinarily-skilled protein chemist.
  • the ⁇ -carbon of an amino acid may be mono- or dimethylated.
  • FGF-21 fusion proteins of the present invention have an in vitro biological activity that is comparable to that of native FGF-21. Although some FGF-21 fusion proteins of the invention may have biological activity lower than that of native FGF-21 as measured in a particular assay, this activity decrease is compensated by the compound's extended half-life and/or lower clearance value and may even be a favorable characteristic for an FGF-21 compound with an extended elimination half-life.
  • the FGF-21 fusion proteins of the present invention can comprise glycosylation sites.
  • Glycosylation is a chemical modification wherein sugar moieties are added to the protein at specific sites. Glycosylation of proteins play a role in ensuring the correct charge, confirmation, and stability of maturing protein and can target the protein to the cell surface and eventual secretion of the protein. Most importantly, glycosylation effects the in vivo clearance rate for many proteins.
  • Sugars can be O-linked or N-linked. Generally, O-linked sugars are added to the hydroxyl-group oxygen of serine and threonine, while N-linked sugars are added to the amide nitrogen of asparagine.
  • the consensus site for N-glycosylation is Asn X1 X2 wherein X1 is any amino acid except Pro and X2 is Ser or Thr.
  • Immunoglobulins are molecules containing polypeptide chains held together by disulfide bonds, typically having two light chains and two heavy chains. In each chain, one domain (V) has a variable amino acid sequence depending on the antibody specificity of the molecule. The other domains (C) have a rather constant sequence common to molecules of the same class.
  • the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, this term refers to an antibody fragment that is obtained by removing the two antigen binding regions (the Fab fragments) from the antibody. One way to remove the Fab fragments is to digest the immunoglobulin with papain protease.
  • the Fc portion is formed from approximately equal sized fragments of the constant region from both heavy chains, which associate through non-covalent interactions and disulfide bonds.
  • the Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the C-terminus of the antibody.
  • the Fc portion can further include one or more glycosylation sites.
  • the amino acid sequence of a representative Fc protein containing a hinge region, CH2 and CH3 domains, and one N-glycosylation site at position 82 is shown below:
  • IgG human immunoglobulin Fc regions with different effector and pharmacokinetic properties: IgG, IgA, IgM, IgD, and IgE.
  • IgG is the most abundant immunoglobulin in serum. IgG also has the longest half-life in serum of any immunoglobulin (23 days). Unlike other immunoglobulins, IgG is efficiently recirculated following binding to an Fc receptor.
  • IgG subclasses G1, G2, G3, and G4 each of which have different effect or functions. These effector functions are generally mediated through interaction with the Fc receptor (Fc ⁇ R) or by binding C1q and fixing complement.
  • Fc ⁇ R binding to Fc ⁇ R can lead to antibody dependent cell mediated cytolysis, whereas binding to complement factors can lead to complement mediated cell lysis.
  • All IgG subclasses are capable of binding to Fc receptors (CD16, CD32, CD64) with G1 and G3 being more effective than G2 and G4.
  • the Fc receptor binding region of IgG is formed by residues located in both the hinge and the carboxy terminal regions of the CH2 domain.
  • the heterologous fusion proteins of the present invention may contain any of the isotypes described above or may contain mutated Fc regions wherein the complement and/or Fc receptor binding functions have been altered.
  • the heterologous fusion proteins of the present invention may contain the entire Fc portion of an immunoglobulin, fragments of the Fc portion of an immunoglobulin, or analogs thereof fused to a FGF-21 compound.
  • the fusion proteins of the present invention can consist of single chain proteins or as multi-chain polypeptides. Two or more Fc fusion proteins can be produced such that they interact through disulfide bonds that naturally form between Fc regions. These multimers can be homogeneous with respect to the FGF-21 compound or they may contain different FGF-21 compounds fused at the N-terminus of the Fc portion of the fusion protein.
  • the Fc or Fc-like region must serve to prolong the in vivo plasma half-life of the FGF-21 compound fused at the C-terminus or N-terminus. Furthermore, the fused FGF-21 compound must retain some biological activity. Biological activity can be determined by in vitro and in vivo methods known in the art. Representative biological assays are described in Examples 1 and 2.
  • the Fc region used for the heterologous fusion proteins of the present invention be derived from an IgG1 or an IgG4 Fc region. It is even more preferable that the heterologous fusion proteins of the present invention contain an Fc portion which is derived from human IgG4, but comprises one or more substitutions compared to the wild-type human sequence.
  • the Fc portion of an immunoglobulin has the meaning commonly given to the term in the field of immunology. Specifically, this term refers to an antibody fragment which does not contain the two antigen binding regions (the Fab fragments) from the antibody.
  • the Fc portion consists of the constant region of an antibody from both heavy chains, which associate through non-covalent interactions and disulfide bonds.
  • the Fc portion can include the hinge regions and extend through the CH2 and CH3 domains to the c-terminus of the antibody.
  • the Fc portion can further include one or more glycosylation sites.
  • heterologous fusion proteins of the present invention are derived from the human IgG4 Fc region because of its reduced ability to bind Fc ⁇ R and complement factors compared to other IgG sub-types.
  • the human IgG4 Fc portion of an immunoglobulin fused to an FGF-21 compound of the present invention comprises the sequence of SEQ ID NO:4 wherein:
  • the IgG4 Fc region which is part of the heterologous fusion proteins of the present invention may contain one or more of the following substitutions that eliminate effector function: substitution of proline for serine at residue 228; substitution of proline for glutamate at residue 233; alanine or valine for phenylalanine at residue 234; and, alanine or glutamate for leucine at residue 235 (EU numbering, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health and Human Services, Bethesda, MD, NIH Publication no. 91-3242 ).
  • residues correspond to positions 11, 16, 17, and 18 in SEQ ID NO:4, respectively. Further, removing the N-linked glycosylation site in the IgG4 Fc region by substituting Ala for Asn at residue 297 (EU numbering) which corresponds to position 80 of SEQ ID NO:4 is another way to ensure that residual effector activity is eliminated in the context of a heterologous fusion protein.
  • the C-terminal lysine residue present in the native molecule may be deleted in the IgG4 derivative Fc portion of the heterologous fusion proteins discussed herein (position 230 of SEQ ID NO:4; deleted lysine referred to as des-K).
  • Fusion proteins expressed in some cell types (such as NSO cells) wherein lysine is encoded by the C-terminal codon are heterogeneous in that a portion of the molecules have lysine as the C-terminal amino acid and a portion have lysine deleted.
  • the deletion is due to protease action during expression in some types of mammalian cells.
  • Fc fusion expression constructs lack a C-terminal codon for lysine.
  • the C-terminal amino acid of the FGF-21 compound discussed herein is fused to the N-terminus of the IgG4 Fc analog portion via a glycine-rich linker (G-rich), designated by L, with the number immediately preceding the L referring to the number of linkers separating the FGF-21 compound from the Fc portion.
  • G-rich glycine-rich linker
  • the length be no longer than necessary to prevent unwanted domain interactions and/or optimize biological activity and/or stability.
  • this linker may be used in the heterologous fusion proteins of the present invention, it is preferred that a single copy of this linker be used to minimize the risk of immunogenicity associated with prolonged and repeated administration.
  • the FGF-21 compound and Fc or HSA portion of the heterologous fusion proteins of the present invention are preferably fused together via 1, 1.5 or 2 repeats of the G-rich peptide linker.
  • the most preferred glycine-rich linker is designated 1L and comprises the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:5).
  • Additional preferred peptide linkers include a linker specified as 1.5L, Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Ser (GEQ ID NO:6); a linker specified as 2L, Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly
  • IgG4 refers to an analog of the human IgG4 Fc sequence specified as SEQ ID NO:4. Substitutions in the IgG4 Fc portion of the heterologous fusion protein are indicated in parenthesis.
  • the wild-type amino acid is specified by its common abbreviation followed by the position number in the context of the entire IgG4 sequence using the EU numbering system followed by the amino acid being substituted at that position specified by its common abbreviation.
  • Preferred FGF-21-Fc-heterlogous fusion proteins of the present invention include the following proteins: I152E/S163E/L118C/A134C-1L-IgG4 (S228P), I152E/S163E/L118C/A134C-1L-IgG4 (S228P, F234A, L235A), I152E/S163E/L118C/A134C-1L-IgG4 (S228P, N297A), I152E/S163E/L118C/A134C-1L-IgG4 (S228P, F234A, L235A, N297A), I152E/S163E/L118C/A134C-1.5L-IgG4 (S228P), I152E/S163E/L118C/A134C-1.5L-IgG4 (S228P, F234A, L235A), I152E/S163E/L118C/A134C
  • More preferred FGF-21-Fc heterologous fusion proteins of the present invention include the following proteins: L118C/A134C-1L-IgG4 (S228P), L118C/A134C-1L-IgG4 (S228P, F234A, L235A), L118C/A134C-1L-IgG4 (S228P, N297A), L118C/A134C-1L-IgG4 (S228P, F234A, L235A, N297A), L118C/A134C-1.5L-IgG4 (S228P), L118C/A134C-1.5L-IgG4 (S228P, F234A, L235A), L118C/A134C-1.5L-IgG4 (S228P, N297A), L118C/A134C-1.5L-IgG4 (S228P, F234A, L235A), L118C/A134C-1.5L-IgG4 (
  • FGF-21-Fc heterologous fusion proteins of the present invention include the following proteins: L118C/A134C/S167A-1L-IgG4 (S228P); L118C/A134C/S167A -1L-IgG4 (S228P, F234A, L235A), L118C/A134C/S167A -1L-IgG4 (S228P, N297A), L118C/A134C/S167A -1L-IgG4 (S228P, F234A, L235A, N297A), L118C/A134C/S167A -1.5L-IgG4 (S228P), L118C/A134C/S167A -1.5L-IgG4 (S228P, F234A, L235A), L118C/A134C/S167A -1.5L-IgG4 (S228P, F234A, L235A), L118C/A134C/S167
  • Heterologous albumin fusion proteins
  • FGF-21 compounds described above can be fused directly or via a peptide linker to albumin or an analog, fragment, or derivative thereof.
  • albumin proteins making up part of the fusion proteins of the present invention can be derived from albumin cloned from any species.
  • human albumin and fragments and analogs thereof are preferred to reduce the risk of the fusion protein being immunogenic in humans.
  • Human serum albumin (HSA) consists of a single non-glycosylated polypeptide chain of 585 amino acids with a formula molecular weight of 66,500. The amino acid sequence of human HSA is: [See Meloun, et al. (1975) FEBS Letters 58:136 ; Behrens, et al. (1975) Fed. Proc. 34:591 ; Lawn, et al.
  • heterologous fusion proteins of the present invention include FGF-21 compounds that are coupled to any albumin protein including fragments, analogs, and derivatives wherein such fusion protein is biologically active and has a longer plasma half-life than the FGF-21 compound alone.
  • the albumin portion of the fusion protein need not necessarily have a plasma half-life equal to that of native human albumin. Fragments, analogs, and derivatives are known or can be generated that have longer half-lives or have half-lives intermediate to that of native human albumin and the FGF-21 compound of interest.
  • the heterologous fusion proteins of the present invention encompass proteins having conservative amino acid substitutions in the FGF-21 compound and/or the Fc or albumin portion of the fusion protein.
  • a "conservative substitution” is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape.
  • Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number carbon and heteroatoms in their side chains differs by no more than about four. They have approximately the same shape when the number of branches in their side chains differs by no more than one.
  • Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Except as otherwise specifically provided herein, conservative substitutions are preferably made with naturally occurring amino acids.
  • amino acid substitutions in the fusion proteins of the present invention can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Furthermore, substitutions can be made based on secondary structure propensity. For example, a helical amino acid can be replaced with an amino acid that would preserve the helical structure. Exemplary substitutions that take various of the foregoing characteristics into consideration in order to produce conservative amino acid changes resulting in silent changes within the present peptides, etc., can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids.
  • the FGF-21 compounds of the present invention may be generated and/or isolated by any means known in the art. Because of the size of the fusion proteins, recombinant methods are preferred such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989 ).
  • Wild-type human IgG4 proteins can be obtained from a variety of sources. For example, these proteins can be obtained from a cDNA library prepared from cells that express the mRNA of interest at a detectable level. Libraries can be screened with probes designed using the published DNA or protein sequence for the particular protein of interest. For example, immunoglobulin light or heavy chain constant regions are described in Adams, et al. (1980) Biochemistry 19:2711-2719 ; Goughet, et al. (1980) Biochemistry 19:2702-2710 ; Dolby, et al. (1980) Proc. Natl. Acad. Sci. USA 77:6027-6031 ; Rice et al. (1982) Proc. Natl. Acad. Sci. USA 79:7862-7862 ; Falkner, et al. (1982) Nature 298:286-288 ; and Morrison, et al. (1984) Ann. Rev. Immunol. 2:239-256 .
  • PCR primers can be designed based on published sequences.
  • the full-length wild-type sequences cloned from a particular library can serve as a template to create the IgG4 Fc analog fragments of the present invention that retain the ability to confer a longer plasma half-life on the FGF-21 compound that is part of the fusion protein.
  • the IgG4 Fc analog fragments can be generated using PCR techniques with primers designed to hybridize to sequences corresponding to the desired ends of the fragment. PCR primers can also be designed to create restriction enzyme sites to facilitate cloning into expression vectors.
  • the gene encoding a fusion protein can then be constructed by ligating DNA encoding a FGF-21 compound in-frame to DNA encoding the IgG Fc proteins described herein.
  • the DNA encoding theFGF-21 compound and IgG4 Fc fragments can be mutated either before ligation or in the context of a cDNA encoding an entire fusion protein. A variety of mutagenesis techniques are well known in the art.
  • the gene encoding the FGF-21 compound and the gene encoding the IgG4 Fc analog protein can also be joined in-frame via DNA encoding a G-rich linker peptide.
  • FGF-21 compounds have a variety of biological activities. FGF-21 is particularly promising as a treatment for non-insulin dependent diabetes mellitus (NIDDM, type 2) as it does not present a risk of hypoglycemia as do present NIDDM treatments. FGF-21 is also contemplated to be a treatment for obesity and metabolic syndrome.
  • NIDDM non-insulin dependent diabetes mellitus
  • FGF-21 fusion proteins of the present invention includes use in the manufacture of a medicament for the treatment of type 2 diabetes, obesity and metabolic syndrome.
  • FGF-21 fusion proteins may be combined with other modifications known in the art to increase FGF-21 half-life and thereby increase the half-life of the compound even further than a fusion protein alone or the other modification method alone.
  • FGF-21 compound also includes pharmaceutically acceptable salts of the compounds described herein.
  • An FGF-21 compound of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt.
  • the FGF-21 fusion proteins of the present invention are particularly suited for parenteral administration, they can be also be delivered orally, by nasal administration, or by inhalation.
  • Parenteral administration can include, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection.
  • the FGF-21 fusion proteins can be administered to the subject in conjunction with an acceptable pharmaceutical carrier, diluent or excipient as part of a pharmaceutical composition for treating the diseases discussed above.
  • the pharmaceutical composition can be a solution or, if administered parenterally, a suspension of the FGF-21.
  • Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the peptide or peptide derivative.
  • Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like.
  • suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, and mannitol.
  • the FGF-21 fusion proteins of the invention may be formulated for administration such that blood plasma levels are maintained in the efficacious range for extended time periods.
  • a “therapeutically effective amount” of a FGF-21 fusion protein is the quantity that results in a desired therapeutic and/or prophylactic effect without causing unacceptable side-effects when administered to a subject.
  • a “desired therapeutic effect” includes one or more of the following: 1) an amelioration of the symptom(s) associated with the disease or condition; 2) a delay in the onset of symptoms associated with the disease or condition; 3) increased longevity compared with the absence of the treatment; and 4) greater quality of life compared with the absence of the treatment.
  • an "effective amount" of a FGF-21 fusion protein for the treatment of type 2 diabetes is the quantity that would result in greater control of blood glucose concentration than in the absence of treatment, thereby resulting in a delay in the onset of diabetic complications such as retinopathy, neuropathy or kidney disease.
  • An "effective amount” of a FGF-21 fusion protein for the prevention of diabetes is the quantity that would delay, compared with the absence of treatment, the onset of elevated blood glucose levels that require treatment with anti-hypoglycaemic drugs such as sulfonyl ureas, thiazolidinediones, insulin and/or bisguanidines.
  • a "therapeutically effective amount" of the FGF-21 fusion protein administered to a subject will also depend on the type and severity of the disease and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs.
  • a typical dose range for the FGF-21 fusion proteins of the present invention will range from about 0.01 mg/kg to about 1000 mg/kg body weight.
  • the dosage ranges from about 0.1 mg/kg to about 100 mg/kg, more preferably from about 1.0 mg/kg to about 10 mg/kg. Most preferably, the dosage is about 1-5 mg/kg body weight.
  • the appropriate dose of a FGF-21 fusion protein administered will result in lowering blood glucose levels and increasing energy expenditure by faster and more efficient glucose utilization, and thus is useful for treating type 2 diabetes, obesity and metabolic syndrome.
  • the frequency of dosing is determined by the plasma half life/clearance of the FGF-21 fusion protein.
  • dosing will be about every 2 days. More preferably, dosing will be about twice weekly. Even more preferably, dosing will be about once weekly. Most preferably, dosing will be about twice a month.
  • FGF-21 fusion proteins are produced in a mammalian cell expression system using HEK293EBNA cells (EdgeBiosystems, Gaiethersburg, MD). FGF-21 fusion proteins are subcloned in the proprietary expression vector representing a modification of commercially available pEAK10, between NheI and XbaI restriction sites in the MCS. The cDNA sequence encoding an FGF-21 fusion protein is fused in frame with the Ig ⁇ leader sequence to enhance secretion of the desired product in the tissue culture media. The expression is driven by the strong viral CMV promoter.
  • HEK293EBNA cells are transiently transfected using a standard transfection reagent such as Fugene (Roche Diagnostics, Indianapolis IN, USA) and the appropriate amount of recombinant plasmid, either as a monolayer or suspension culture, at the adequate cell density. Cells are incubated at 37°C and 5% CO 2 , in serum free media, and collections are made every day for 5 days. Typically the expression level in the HEK293EBNA suspension culture is - 30 mg/L. The expression of an FGF-21 fusion protein in mammalian cells yields the natural N-terminal sequence, HPIP, i.e. without a methionine residue at the N-terminus
  • Fractions are analyzed by SDS PAGE, pooled and concentrated by Millipore 30K Amicon ultra centrifugal filter devices and filter sterilized using a 0.22 ⁇ m MILLEX-GV filter (Millipore SLGVR25CS). The final concentration is determined by absorbance at 280nm (scatter corrected). MALDI and N-terminal sequence are used to confirm the protein.
  • fraction pool is concentrated with Millipore 30K Amicon ultra centrifugal filter devices ( UFC903024) and loaded onto a 26/60 Superdex 200 column ( Amersham Bioscience AB, Uppsala, Sweden), equilibrated in PBS, pH7.4. Fractions are analyzed by SDS-PAGE, pooled, concentrated and filter sterilized. The final concentration is determined by absorbance at 280nm (scatter corrected). MALDI and N-terminal sequence are used to confirm the protein.
  • yeast such as Pichia pastoris, Pichia methanolica or Saccharomyces cerevisiae.
  • yeast such as Pichia pastoris, Pichia methanolica or Saccharomyces cerevisiae.
  • AOX1 alcohol oxidase
  • vectors that use the promoter from the GAP gene are available for high level constitutive expression.
  • the multi-copy Pichia expression vectors allow one to obtain strains with multiple copies of the gene of interest integrated into the genome. Increasing the number of copies of the gene of interest in a recombinant Pichia strain can increase protein expression levels.
  • 3T3-L1 cells are obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells are cultured in growth medium (GM) containing 10% iron-enriched fetal bovine serum in Dulbecco's modified Eagle's medium. For standard adipocyte differentiation, two days after cells reached confluency (referred as day 0), cells are exposed to differentiation medium (DM) containing 10% fetal bovine serum, 10 ⁇ g/ml of insulin, 1 ⁇ M dexamethasone, and 0.5 ⁇ M isobutylmethylxanthine, for 48 h. Cells then are maintained in post differentiation medium containing 10% fetal bovine serum, and 10 ⁇ g/ml of insulin.
  • GM growth medium
  • DM differentiation medium
  • Glucose Transport Assay-- Hexose uptake as assayed by the accumulation of 0.1 mM 2-deoxy-D-[ 14 C]glucose, is measured as follows: 3T3-L1 adipocytes in 12-well plates are washed twice with KRP buffer (136 mM NaCl, 4.7 mM KCI, 10mM NaPO 4 , 0.9 mM CaCl 2 , 0.9 mM MgSO 4 , pH 7.4) warmed to 37 °C and containing 0.2% BSA, incubated in Leibovitz's L-15 medium containing 0.2% BSA for 2 h at 37°C in room air, washed twice again with KRP containing, 0.2% BSA buffer, and incubated in KRP, 0.2% BSA buffer in the absence (Me 2 SO only) or presence of wortmannin for 30 min at 37 °C in room air.
  • KRP buffer 136 mM NaCl, 4.7 mM KCI,
  • Insulin is then added to a final concentration of 100 nM for 15 min, and the uptake of 2-deoxy-D-[ 14 C]glucose is measured for the last 4 min.
  • Nonspecific uptake measured in the presence of 10 ⁇ M cytochalasin B, is subtracted from all values. Protein concentrations are determined with the Pierce bicinchoninic acid assay. Uptake is measured routinely in triplicate or quadruplicate for each experiment.
  • In vitro potency (EC 50 ) is compared to the in vitro activity of wild-type FGF-21.
  • the in vitro potency of FGF-21 fusion proteins of the present invention is compared to wild-type FGF-21 in Table 1. As indicated in Table 1, the FGF-21 fusion proteins of the present invention have reduced in vitro potency to various degrees compare to wild-type FGF-21. However, the decrease in in vitro potency is likely compensated for with the increase in time extension (plasma half life) of the FGF-21 fusion protein.
  • the Ob / ob mouse model is an animal model for hyperglycemia, insulin resistance and obesity.
  • Male ob / ob mice are used to monitor plasma glucose levels and triglyceride levels after treatment with FGF-21 fusion proteins compared to FGF-21 alone.
  • the test groups of male ob / ob mice (7 weeks old) are: (1) s.c.
  • FGF-21-Fc fusion protein is administered s.c. in 0.1 ml.
  • Blood glucose levels are measured daily for 7 days, 1 hour post dosing, using a standard protocol.
  • the extended time action of FGF-21-Fc fusion proteins is indicated in Table 2 where a single dose on day 0 lowers blood glucose levels for 6 days. TABLE 2 Treatment Blood Glucose Levels in ob / ob mice (mg/dl) Days of Treatment 0 1 2 3 4 5 6 7 Veh. Ctl.
  • FGF-21-Fc fusion proteins FGF-21 C terminus is fused to the N-terminus of an IgG4 Fc, without a linker peptide.
  • male ob / ob mice are used to monitor plasma glucose levels after a single treatment with a FGF-21 fusion protein fused with a linker peptide, compared to continuous infusion of FGF-21 alone.
  • the test groups of male ob / ob mice (7 weeks old) are: (1) vehicle control (0.9% NaCl) by continuous infusion for seven days (Alzet pumps 1007D, 100mcl, 0.5mcl/h); (2) FGF-21, 3.4nM by continuous infusion for seven days; (3) FGF-21-L-Fc fusion protein, 3.4nM; administered s.c.
  • Fc-L-FGF-21 fusion protein 3.4 nM administered s . c . in 0.1 ml on Day 0 only
  • FGF-21-L-HSA fusion protein 3.4 nM administered s.c. in 0.1 ml on Day 0 only.
  • L is the peptide linker, (Gly-Gly-Gly-Gly-Ser) 3 , in the above fusion proteins
  • the animals of groups (1) and (2) are dosed by continuous infusion for 7 days and groups (3) through (5) are dosed on day 0 only. Blood glucose levels are measured daily for 7 days, 1 hour post dosing, using a standard protocol. The superior extended time action of FGF-21 fusion proteins is demonstrated in Table 3, where a single dose on day 0 lowers blood glucose levels for 7 days.
  • FGF-21 fusion proteins are administered by intravenous (IV) or subcutaneous (SC) routes at a dose of 0.4 mg/kg to CD-1 mice. The animals are bled at various times between 0 and 336 hours after dosing. Plasma is collected from each sample and analyzed by radioimmunoassay. Pharmacokinetic parameters are calculated using model-dependent (IV data) and independent (SC data) methods (WinNonlin Pro) and are reported in Table 5 below.
  • IV administration the FGF-21-Fc fusion protein has an elimination half-life of approximately 53.9 hours compared to an elimination half-life of 0.5 hours for native FGF-21.
  • the FGF-21-Fc fusion protein has an elimination half-life of approximately 24 hours compared to an elimination half-life of 0.6 hours for native FGF-21.
  • the FGF-21-Fc fusion protein demonstrates prolonged time action when compared to native FGF-21.
  • the FGF-21-HSA fusion protein has an elimination half-life of approximately 14.3 hours compared to an elimination half-life of 0.5 hours for native FGF-21.
  • SC administration the FGF-21-HSA fusion protein has an elimination half-life of approximately 8 hours compared to an elimination half-life of 0.6 hours for native FGF-21.
  • both routes of administration the FGF-21-HSA fusion protein demonstrates prolonged time action when compared to native FGF-21.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Diabetes (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Obesity (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Child & Adolescent Psychology (AREA)
  • Emergency Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP09150627A 2004-05-13 2005-05-02 FGF-21 fusion proteins Withdrawn EP2194064A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US57090804P 2004-05-13 2004-05-13
EP05742237A EP1751184B1 (en) 2004-05-13 2005-05-02 Fgf-21 fusion proteins

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
EP05742237.0 Division 2005-05-02
WOPCT/US2005/015111 Previously-Filed-Application 2005-05-02
EP05742237A Division EP1751184B1 (en) 2004-05-13 2005-05-02 Fgf-21 fusion proteins

Publications (1)

Publication Number Publication Date
EP2194064A1 true EP2194064A1 (en) 2010-06-09

Family

ID=92541399

Family Applications (2)

Application Number Title Priority Date Filing Date
EP05742237A Expired - Lifetime EP1751184B1 (en) 2004-05-13 2005-05-02 Fgf-21 fusion proteins
EP09150627A Withdrawn EP2194064A1 (en) 2004-05-13 2005-05-02 FGF-21 fusion proteins

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP05742237A Expired - Lifetime EP1751184B1 (en) 2004-05-13 2005-05-02 Fgf-21 fusion proteins

Country Status (12)

Country Link
US (1) US7576190B2 (pl)
EP (2) EP1751184B1 (pl)
JP (1) JP2008506635A (pl)
AT (1) ATE444306T1 (pl)
CA (1) CA2565300A1 (pl)
DE (1) DE602005016917D1 (pl)
DK (1) DK1751184T3 (pl)
ES (1) ES2332100T3 (pl)
PL (1) PL1751184T3 (pl)
PT (1) PT1751184E (pl)
SI (1) SI1751184T1 (pl)
WO (1) WO2005113606A2 (pl)

Families Citing this family (103)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7459540B1 (en) 1999-09-07 2008-12-02 Amgen Inc. Fibroblast growth factor-like polypeptides
US8981061B2 (en) 2001-03-20 2015-03-17 Novo Nordisk A/S Receptor TREM (triggering receptor expressed on myeloid cells) and uses thereof
US9453251B2 (en) 2002-10-08 2016-09-27 Pfenex Inc. Expression of mammalian proteins in Pseudomonas fluorescens
US8603824B2 (en) 2004-07-26 2013-12-10 Pfenex, Inc. Process for improved protein expression by strain engineering
EA011390B1 (ru) 2004-09-02 2009-02-27 Эли Лилли Энд Компани Мутантные белки (мутеины) фактора роста фибробластов 21
GB0426146D0 (en) 2004-11-29 2004-12-29 Bioxell Spa Therapeutic peptides and method
KR100888022B1 (ko) * 2006-12-21 2009-03-09 재단법인 목암생명공학연구소 면역글로불린 Ｆｃ와 인간 아포리포단백질（ａ）크링글절편의 융합단백질 ＬＫ8­Ｆｃ
CA2682147C (en) 2007-03-30 2017-08-08 Ambrx, Inc. Modified fgf-21 polypeptides and their uses
JP5444553B2 (ja) 2007-04-27 2014-03-19 フェネックス インコーポレイテッド 微生物宿主を迅速にスクリーニングして、異種タンパク質発現の収率および／または質が改善されている特定の株を同定する方法
US9580719B2 (en) 2007-04-27 2017-02-28 Pfenex, Inc. Method for rapidly screening microbial hosts to identify certain strains with improved yield and/or quality in the expression of heterologous proteins
CA2693504A1 (en) 2007-08-03 2009-02-12 Eli Lilly And Company Treatment for obesity
AU2008304748C1 (en) 2007-09-26 2014-06-12 Chugai Seiyaku Kabushiki Kaisha Modified antibody constant region
EP2296690B1 (en) 2008-06-04 2016-11-30 Amgen, Inc Fgf21 mutants and uses thereof
JP5878757B2 (ja) 2008-10-10 2016-03-08 アムジエン・インコーポレーテツド Ｆｇｆ２１変異体およびその使用
BRPI1007313A2 (pt) 2009-01-23 2016-02-10 Novo Nordisk As derivados de fgf21 com ligante de albumina a-b-c-d-e e seu uso.
EP2427207B1 (en) 2009-05-05 2017-08-16 Amgen, Inc Fgf21 mutants and uses thereof
HRP20240135T1 (hr) * 2009-05-05 2024-04-12 Amgen Inc. Fgf21 mutanti i njihove upotrebe
WO2011154349A2 (en) 2010-06-08 2011-12-15 Novo Nordisk A/S Fgf21 analogues and derivatives
US8324160B2 (en) * 2009-06-17 2012-12-04 Amgen Inc. Chimeric polypeptides and uses thereof
MX368790B (es) * 2009-10-15 2019-10-16 Genentech Inc Factores de crecimiento de fibroblasto quimericos con especificidad de receptor alterada.
EP2679234A3 (en) 2009-12-02 2014-04-23 Amgen Inc. Binding proteins that bind to human FGFR1C, human beta-klotho and both human FGFR1C and human beta-klotho
UA109888C2 (uk) 2009-12-07 2015-10-26 ІЗОЛЬОВАНЕ АНТИТІЛО АБО ЙОГО ФРАГМЕНТ, ЩО ЗВ'ЯЗУЄТЬСЯ З β-КЛОТО, РЕЦЕПТОРАМИ FGF І ЇХНІМИ КОМПЛЕКСАМИ
EP2460527A1 (en) 2010-01-21 2012-06-06 Sanofi Pharmaceutical composition for treating a metabolic syndrome
US10745467B2 (en) 2010-03-26 2020-08-18 The Trustees Of Dartmouth College VISTA-Ig for treatment of autoimmune, allergic and inflammatory disorders
BR112012024565B1 (pt) 2010-03-26 2022-02-08 Trustees Of Dartmouth College Proteína de fusão vista imunossupressora multimérica isolada ou recombinante e composição
US20150231215A1 (en) 2012-06-22 2015-08-20 Randolph J. Noelle VISTA Antagonist and Methods of Use
CA2796055A1 (en) 2010-04-15 2011-10-20 Amgen Inc. Human fgf receptor and .beta.-klotho binding proteins
CA2796459C (en) 2010-04-16 2016-05-24 Salk Institute For Biological Studies Methods for treating metabolic disorders using fgf-1
EP2595647A1 (en) * 2010-07-20 2013-05-29 Novo Nordisk A/S N-terminal modified fgf21 compounds
JP2014500248A (ja) * 2010-11-05 2014-01-09 コヴェックス・テクノロジーズ・アイルランド・リミテッド 抗糖尿病化合物
US9023791B2 (en) * 2010-11-19 2015-05-05 Novartis Ag Fibroblast growth factor 21 mutations
CN110551223A (zh) 2011-06-28 2019-12-10 英伊布里克斯有限合伙公司 丝氨酸蛋白酶抑制蛋白融合多肽及其使用方法
WO2013006486A2 (en) 2011-07-01 2013-01-10 Ngm Biopharmaceuticals, Inc. Compositions, uses and methods for treatment of metabolic disorders and diseases
EP2548570A1 (en) 2011-07-19 2013-01-23 Sanofi Pharmaceutical composition for treating a metabolic syndrome
UY34346A (es) * 2011-09-26 2013-04-30 Novartis Ag Proteínas de fusión para tratar trastornos metabólicos
JP6165168B2 (ja) 2011-12-22 2017-07-19 ファイザー・インク 抗糖尿病化合物
JP6400480B2 (ja) 2012-02-15 2018-10-03 ノヴォ ノルディスク アー／エス ペプチドグリカン認識タンパク質１に結合する抗体
US9550830B2 (en) 2012-02-15 2017-01-24 Novo Nordisk A/S Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1)
SMT201700488T1 (it) 2012-02-15 2017-11-15 Novo Nordisk As Anticorpi che legano e bloccano il recettore d'innesco espresso sulle cellule mieloidi-1 (trem-1)
US9475856B2 (en) 2012-03-02 2016-10-25 New York University Chimeric FGF21 proteins with enhanced binding affinity for β-klotho for the treatment of type II diabetes, obesity, and related metabolic disorders
US9474785B2 (en) 2012-06-07 2016-10-25 New York University Chimeric fibroblast growth factor 19 proteins and methods of use
US9657075B2 (en) 2012-06-07 2017-05-23 New York University Chimeric fibroblast growth factor 23 proteins and methods of use
US9464126B2 (en) 2012-06-07 2016-10-11 New York University Chimeric fibroblast growth factor 21 proteins and methods of use
CA2871656A1 (en) 2012-06-11 2013-12-19 Eli Lilly And Company Fibroblast growth factor 21 variants
TWI513705B (zh) 2012-06-11 2015-12-21 Lilly Co Eli 纖維母細胞生長因子２１蛋白質
DK3421486T5 (da) * 2012-06-22 2024-09-16 The Trustees Of Darthmouth College Nye Vista-IG-konstruktioner og anvendelse af Vista-IG til behandling af autoimmune, allergiske og inflammatoriske lidelser
US9890215B2 (en) 2012-06-22 2018-02-13 King's College London Vista modulators for diagnosis and treatment of cancer
JP6368308B2 (ja) 2012-09-07 2018-08-01 トラスティーズ・オブ・ダートマス・カレッジ 癌の診断および治療のためのｖｉｓｔａ調節剤
US9290557B2 (en) 2012-11-28 2016-03-22 Ngm Biopharmaceuticals, Inc. Compositions comprising variants and fusions of FGF19 polypeptides
HK1214832A1 (zh) 2012-11-28 2016-08-05 恩格姆生物制药公司 用於代謝病症和疾病治療的組合物和方法
US9273107B2 (en) 2012-12-27 2016-03-01 Ngm Biopharmaceuticals, Inc. Uses and methods for modulating bile acid homeostasis and treatment of bile acid disorders and diseases
MX383664B (es) 2012-12-27 2025-03-14 Ngm Biopharmaceuticals Inc Uso de un péptido para modular la homeostasis de los ácidos biliares o tratamiento de una enfermedad relacionada con los ácidos biliares.
US9550820B2 (en) 2013-02-22 2017-01-24 New York University Chimeric fibroblast growth factor 23/fibroblast growth factor 19 proteins and methods of use
EP3057605A1 (en) 2013-10-18 2016-08-24 Novartis AG Methods of treating diabetes and related disorders
AU2014340241B2 (en) 2013-10-21 2018-11-01 Salk Institute For Biological Studies Mutated fibroblast growth factor (FGF) 1 and methods of use
WO2015065897A1 (en) 2013-10-28 2015-05-07 Ngm Biopharmaceuticals, Inc. Cancer models and associated methods
RS63295B1 (sr) 2013-12-24 2022-06-30 Janssen Pharmaceutica Nv Anti-vista antitela i fragmenti
US11014987B2 (en) 2013-12-24 2021-05-25 Janssen Pharmaceutics Nv Anti-vista antibodies and fragments, uses thereof, and methods of identifying same
PT3097122T (pt) 2014-01-24 2020-07-21 Ngm Biopharmaceuticals Inc Anticorpos de ligação de domínio 2 de beta klotho e métodos de utilização dos mesmos
US20170065678A1 (en) 2014-03-11 2017-03-09 Novartis Ag Methods of treating metabolic disorders associated with lipodystrophies and defects in insulin production or signaling
US10398758B2 (en) 2014-05-28 2019-09-03 Ngm Biopharmaceuticals, Inc. Compositions comprising variants of FGF19 polypeptides and uses thereof for the treatment of hyperglycemic conditions
MX389695B (es) 2014-06-11 2025-03-20 Kathy A Green Uso de agonistas y antagonistas vista para suprimir o aumentar la inmunidad humoral.
WO2015195509A2 (en) 2014-06-16 2015-12-23 Ngm Biopharmaceuticals, Inc. Methods and uses for modulating bile acid homeostasis and treatment of bile acid disorders and diseases
EP3160496B1 (en) * 2014-06-24 2021-03-03 Novo Nordisk As Mic-1 fusion proteins and uses thereof
EP3172232B1 (en) 2014-07-17 2023-12-27 Novo Nordisk A/S Site directed mutagenesis of trem-1 antibodies for decreasing viscosity.
CN114129709A (zh) 2014-10-23 2022-03-04 恩格姆生物制药公司 包含肽变异体的药物组合物及其使用方法
SMT202100388T1 (it) 2014-10-24 2021-09-14 Bristol Myers Squibb Co Polipeptidi fgf-21 modificati e loro usi
US10434144B2 (en) 2014-11-07 2019-10-08 Ngm Biopharmaceuticals, Inc. Methods for treatment of bile acid-related disorders and prediction of clinical sensitivity to treatment of bile acid-related disorders
JP2018505911A (ja) 2014-12-05 2018-03-01 イミュネクスト，インコーポレーテッド 推定上のｖｉｓｔａ受容体としてのｖｓｉｇ８の同定と、ｖｉｓｔａ／ｖｓｉｇ８調節剤を産生するためのその使用
AR103246A1 (es) 2014-12-23 2017-04-26 Novo Nordiks As Derivados de fgf21 y sus usos
KR20160088656A (ko) * 2015-01-16 2016-07-26 주식회사유한양행 지속형 fgf21 융합 단백질 및 이를 포함하는 약학적 조성물
WO2016207717A1 (en) 2015-06-24 2016-12-29 Janssen Pharmaceutica Nv Anti-vista antibodies and fragments
WO2017019957A2 (en) 2015-07-29 2017-02-02 Ngm Biopharmaceuticals, Inc. Binding proteins and methods of use thereof
WO2017075260A1 (en) 2015-10-30 2017-05-04 Salk Institute For Biological Studies Treatment of steroid-induced hyperglycemia with fibroblast growth factor (fgf) 1 analogs
JP6728352B2 (ja) 2015-11-09 2020-07-22 エヌジーエム バイオファーマシューティカルス，インコーポレーテッド 胆汁酸に関係した障害の治療方法
US10899836B2 (en) 2016-02-12 2021-01-26 Janssen Pharmaceutica Nv Method of identifying anti-VISTA antibodies
CN109789201B (zh) 2016-04-15 2023-06-16 伊穆奈克斯特股份有限公司 抗人vista抗体及其用途
CN109414471A (zh) 2016-05-24 2019-03-01 诺和诺德股份有限公司 Mic-1化合物及其用途
CN106279437B (zh) 2016-08-19 2017-10-31 安源医药科技（上海）有限公司 高糖基化人凝血因子viii融合蛋白及其制备方法与用途
CN107759697B (zh) 2016-08-19 2023-03-24 安源医药科技（上海）有限公司 制备融合蛋白的方法
US11123438B2 (en) 2016-08-19 2021-09-21 Ampsource Biopharma Shanghai Inc. Linker peptide for constructing fusion protein
EP3503882A4 (en) 2016-08-26 2020-07-29 NGM Biopharmaceuticals, Inc. METHOD FOR TREATING FIBROBLAST GROWTH FACTOR-19-MEDIATED CARCINOMAS AND TUMORS
EP4470551A3 (en) * 2017-03-14 2025-02-26 Sunshine Lake Pharma Co., Ltd. Dual-target fusion proteins comprising the fc portion of an immunoglobulin
AR111341A1 (es) 2017-03-23 2019-07-03 Hanmi Pharm Ind Co Ltd Un conjugado del análogo de insulina con afinidad reducida para el receptor de insulina y uso del mismo
TWI710377B (zh) 2017-05-23 2020-11-21 丹麥商諾佛 儂迪克股份有限公司 Ｍｉｃ－１化合物及其用途
MX2020002206A (es) 2017-09-08 2020-07-20 Bristol Myers Squibb Co Factor de crecimiento de fibroblastos 21 (fgf-21) modificado para usarse en metodos para tratar la esteatohepatitis no alcoholica (nash).
US20200376080A1 (en) 2017-12-22 2020-12-03 Novartis Ag Methods of treating metabolic disorders with fgf21 variants
CN111727055B (zh) * 2018-02-06 2024-06-25 温哥华生物技术有限公司 GHR-106单克隆抗体作为GnRH拮抗剂的用途
AU2019218147B2 (en) 2018-02-08 2023-06-08 Sunshine Lake Pharma Co., Ltd. FGF21 variant, fusion protein and application thereof
EP3774902A1 (en) 2018-04-02 2021-02-17 Bristol-Myers Squibb Company Anti-trem-1 antibodies and uses thereof
CA3104686A1 (en) 2018-07-03 2020-01-09 Bristol-Myers Squibb Company Fgf-21 formulations
CN111195349B (zh) * 2018-11-16 2023-02-24 鲁南制药集团股份有限公司 一种代谢调节融合蛋白的冻干粉制剂
CN111662373B (zh) 2019-03-05 2024-05-14 广东东阳光药业股份有限公司 一种多肽分子及其应用
CN114853908B (zh) * 2019-05-16 2024-06-07 浙江道尔生物科技有限公司 一种治疗代谢疾病的融合蛋白
US11542309B2 (en) 2019-07-31 2023-01-03 Salk Institute For Biological Studies Fibroblast growth factor 1 (FGF1) mutant proteins that selectively activate FGFR1B to reduce blood glucose
IL294534A (en) 2020-01-08 2022-09-01 Bristol Myers Squibb Co Fgf-21 conjugate formulations
CN115322794B (zh) 2020-01-11 2025-09-19 北京质肽生物医药科技有限公司 Glp-1和fgf21的融合蛋白的缀合物
US11981718B2 (en) 2020-05-27 2024-05-14 Ampsource Biopharma Shanghai Inc. Dual-function protein for lipid and blood glucose regulation
EP4230216A4 (en) * 2020-07-24 2025-02-19 Jiangsu Gensciences Inc. Insulin-fc fusion protein and application thereof
JP2023538533A (ja) 2020-08-07 2023-09-08 ブリストル－マイヤーズ スクイブ カンパニー 線維症の処置のための、ｃｃｒ２／５拮抗剤と組み合わせたｆｇｆ２１
US20240123031A1 (en) 2020-11-25 2024-04-18 Bristol-Myers Squibb Company Methods of treating liver diseases
CA3224743A1 (en) 2021-07-14 2023-01-19 Beijing Ql Biopharmaceutical Co., Ltd. Fusion polypeptides for metabolic disorders
WO2024132147A1 (en) 2022-12-22 2024-06-27 Lifearc Galanin-2 receptor agonists

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018921A2 (en) * 1998-09-30 2000-04-06 Chiron Corporation Synthetic peptides having fgf receptor affinity
WO2001036640A2 (en) * 1999-11-18 2001-05-25 Chiron Corporation Human fgf-21 gene and gene expression products
WO2001072957A2 (en) * 2000-03-31 2001-10-04 Nobuyuki Itoh Fibroblast growth factor-like molecules and uses thereof
WO2004110472A2 (en) * 2003-06-12 2004-12-23 Eli Lilly And Company Fusion proteins

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US606830A (en) 1898-07-05 Insulator
US528582A (en) 1894-11-06 Can-opener
US606805A (en) 1898-07-05 Can-opener
US635882A (en) 1898-11-02 1899-10-31 Henry Torrance Jr Refrigerating system.
JPH10503371A (ja) 1994-07-29 1998-03-31 スミスクライン・ビーチャム・パブリック・リミテッド・カンパニー 新規化合物
US7459540B1 (en) 1999-09-07 2008-12-02 Amgen Inc. Fibroblast growth factor-like polypeptides
US6716626B1 (en) * 1999-11-18 2004-04-06 Chiron Corporation Human FGF-21 nucleic acids
BR0116024A (pt) * 2000-12-07 2005-12-13 Lilly Co Eli Proteìna de fusão heteróloga e uso da mesma

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018921A2 (en) * 1998-09-30 2000-04-06 Chiron Corporation Synthetic peptides having fgf receptor affinity
WO2001036640A2 (en) * 1999-11-18 2001-05-25 Chiron Corporation Human fgf-21 gene and gene expression products
WO2001072957A2 (en) * 2000-03-31 2001-10-04 Nobuyuki Itoh Fibroblast growth factor-like molecules and uses thereof
WO2004110472A2 (en) * 2003-06-12 2004-12-23 Eli Lilly And Company Fusion proteins

Also Published As

Publication number Publication date
PL1751184T3 (pl) 2010-02-26
EP1751184A2 (en) 2007-02-14
EP1751184B1 (en) 2009-09-30
DE602005016917D1 (de) 2009-11-12
CA2565300A1 (en) 2005-12-01
US7576190B2 (en) 2009-08-18
ES2332100T3 (es) 2010-01-26
WO2005113606A2 (en) 2005-12-01
DK1751184T3 (da) 2009-11-23
SI1751184T1 (sl) 2010-01-29
US20070237768A1 (en) 2007-10-11
PT1751184E (pt) 2009-11-10
JP2008506635A (ja) 2008-03-06
ATE444306T1 (de) 2009-10-15
WO2005113606A3 (en) 2006-05-04

Similar Documents

Publication Publication Date Title
EP1751184B1 (en) Fgf-21 fusion proteins
US20250084144A1 (en) Dual-target fusion proteins comprising fgf21 and glp-1, connected through a fc portion of an immunoglobulin
JP4809352B2 (ja) 線維芽細胞成長因子２１の突然変異タンパク質
JP6182600B2 (ja) 線維芽細胞増殖因子２１タンパク質
CN110028587B (zh) 用于调节血糖和脂质的增效型双功能蛋白
JP6181752B2 (ja) 線維芽細胞増殖因子２１変異体
US20070265200A1 (en) Glycol Linked Fgf-21 Compounds
EP2292651A1 (en) Methods and compositions for the prevention and treatment of anemia
CN106559984A (zh) 用于治疗糖尿病的包含长效胰岛素类似物缀合物和长效促胰岛素肽缀合物的组合物
JP2007536902A (ja) Ｇｌｐ−１アナログ複合タンパク質
EA005584B1 (ru) Слитые белки glp-1
CN110172103B (zh) GLP-1类似物-Fc融合蛋白及其制备方法和用途
KR20220157910A (ko) Gdf15 변이체 및 glp-1 수용체 작용제를 포함하는 병용 투여용 조성물
AU2002326742A1 (en) KGF polypeptide compositions
HK40015284B (en) Dual-target fusion proteins comprising the fc portion of an immunoglobulin
HK40015284A (en) Dual-target fusion proteins comprising the fc portion of an immunoglobulin
HK1045160A (en) Expression and export of anti-obesity proteins as fc fusion proteins

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AC Divisional application: reference to earlier application

Ref document number: 1751184

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR

AKY No designation fees paid
REG Reference to a national code

Ref country code: DE

Ref legal event code: 8566

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20101210